Alzheimer's disease (AD) is a very widespread neurodegenerative form of dementia which affects a high percentage of the world's population over 70 years old, with a ratio of 1:2 between men and women. The incidence of AD is constantly increasing, as the more common non-genetic form begins at around the age of 70-75, and life expectancy is increasing due to higher standards of living combined with the progress of medicine and pharmacology. The disease also exists as a genetically transmitted form associated with mutations in some loci of chromosomes 14, 19 and 21 (typical of persons suffering from Down's syndrome). Early onset of the familial form of AD takes place at around 50 years old, and causes brain degeneration followed by death in 2-3 years. The late-onset form of the disease also gives rise to brain degeneration and death, but in a period of 10 or more years.
The brain presents a large number of plaques in the interneuronal spaces and typical neurofibrillary tangles in the neurons, especially those of the cerebral cortex, hippocampus and amygdala, and in other parts of the brain with cognitive functions. Amyloid plaques, also known as senile plaques, are polymers of the peptide beta-amyloid (Aβ), which derives from a larger protein: beta-amyloid precursor protein (APP). APP is a member of the highly conserved superfamily of transmembrane glycoproteins.
In the last decade, numerous studies have been conducted by scientists engaged in research into AD with a view to understanding its etiology, especially as regards the molecular mechanisms. Research into the molecular components and their regulation can clarify their therapeutic and diagnostic prospects. Attention has focused on presenilins, whose role in processing APP, and therefore in producing Aβ, appears to be highly significant. It has been demonstrated that these proteins, presenilin 1 (PS1) and presenilin 2 (PS2), present an enzymatic activity or regulate the activities of other enzymes, namely secretases, which cleave APP into normally degradable catabolites (alpha-secretase) or into peptide Aβ (beta- and gamma-secretase). In familial AD, mutations of the genes coding for PS1 and PS2 lead to excessive production of Aβ and accumulation of isoform Aβ-42 in particular, which is highly amyloidogenic. Recently, experiments on PS1 knockout mice evidenced a great reduction in gamma-secretase activity, demonstrating that PS1, as well as belonging to the gamma-secretase complex, is also mainly responsible for the production and accumulation of Aβ. As regards beta-secretase, it is believed that the product of the BACE gene can perform beta-secretase cleavage alone.
The development of clinically useful gamma- and beta-secretase inhibitors could become a crucial weapon against Alzheimer's disease, and is currently one of the most exciting competitions in neuroscience. It has been clearly demonstrated that the activity of PS1 cannot be wholly suppressed, because this protein is needed to process transduction factor Notch-1, a crucial factor for the maturity of many stem cells, such as those involved in erythropoiesis.
Regulation of gene expression by means of DNA methylation can be successfully studied in a cell culture system able to express the genes involved in AD. As a result of our studies, a very interesting indication has been found in the regulatory mechanisms involved in aging, which consists of a gradual, global increase in DNA hypomethylation in the elderly, and homocysteine accumulation observed in patients suffering from senile dementia. Homocysteine accumulation and DNA hypomethylation are metabolically correlated, because the failure of homocysteine to be converted to methionine reverses the metabolism towards synthesis of S-adenosylhomocysteine, which is known to be a strong DNA-methyltransferase inhibitor, and therefore induces DNA hypomethylation. In accordance with the well-established theory that many genes are expressed when the cytosines of specific sequences are demethylated, this biochemical pattern can lead to expression of unexpressed genes and overexpression of normally expressed genes. This may be the case with AD, because the overexpression of PS1, namely gamma-secretase, may discontinuously exceed alpha-secretase activity, and therefore produce the peptide Aβ, which accumulates, and can cause the disease after many years. A further indication of the possible role of DNA methylation in AD is the finding that AD patients present much lower post mortem levels of methyl donors in the brain. Lower availability of S-adenosylmethionine (SAM) could easily lead to altered or increased expression of the genes involved in APP metabolism, eventually producing an accumulation of peptide Aβ in the senile plaques.
Preliminary experiments have been performed on a neuroblastoma line (SK-N-SH) which expresses APP, PS1, PS2, BACE, alpha-secretase, the other components of gamma-secretase, and Notchl. The cultures were treated with a culture medium deprived of folate, vitamin B12 and vitamin B6 (in order to alter the metabolism of homocysteine), to which SAM was added at various concentrations (to balance the effects of vitamin deprivation). We found an increase in PS1 and BACE expression in the vitamin B-deprived medium, and a marked reduction in PS1 and BACE expression after the administration of SAM. In experiments conducted with HpaII/PCR on APP and PS1 promoters, we found a major difference in the methylation of one of the CpG sites of the PS1 promoter. We concluded that the PS1 gene can be partly silenced by administration of exogenous SAM. The administration of SAM can reduce PS1 expression, restoring the metabolic balance in favour of alpha-secretase. Experiments have also been conducted with transgenic mice of the strain TgCRND8, and corresponding controls; these mice are characterised by the presence of the human mutated APP gene, and can therefore develop amyloid plaques in a short time. These animals were fed on a complete diet or a diet lacking in the vitamins of the B group; once again, as in the cells, an increase in PS1 and BACE expression was observed.
In both the experimental models, the alteration of gene expression had the effect of increasing gamma- and beta-secretase activities with consequent overproduction of Aβ, which accumulated to form senile plaques more rapidly than in the animals treated with the control diet.
The data summarised above have been reported in the following publications:    Fuso A., Nicolia V., Cavallaro R. A., Ricceri L., D'Anselmi F., Coluccia P., Calamandrei G. and Scarpa S. 2008. B-Vitamin Deprivation Induces Hyperhomocysteinemia and Brain S-adenosylhomocsyteine, Depletes Brain S-adenosylmethionine, and Enhances PS1 and BACE Expression and Amyloid-β Deposition in Mice. Mol. Cell. Neurosci. 37: 731-746.    Fuso A., Cavallaro R. A., Zampelli A., D'Anselmi F., Piscopo P., Confaloni A. and Scarpa S. 2007. γ-secretase is differentially modulated by alterations of Homocysteine cycle in neuroblastoma and glioblastoma cells. J. Alz. Dis. 11: 275-290.    Cavallaro R. A., Fuso A., D'Anselmi F. and Scarpa S. 2006. The effect of S-adenosylmethyonine on CNS gene expression studied by cDNA mycroarrays analysis. J. Alz. Disease. 9: 415-419.    Scarpa S., Cavallaro R. A., D'Anselmi F. and Fuso A. 2006. Gene silencing through methylation: an epigenetic intervention on Alzheimer Disease. J. Alz. Disease. 9: 407-414.    Fuso A., Seminara L., Cavallaro R. A., D'Anselmi F. and Scarpa S. 2004. Homocysteine/S-adenosylmethionine Cycle Alterations Unbalance DNA Methylation Status with Consequent Up-regulation of Beta-amyloid Prmnotinn. Mnl. cell. Nelirnri. 28(1):195-204.    Scarpa S., Fuso A., D'Anselmi F., Cavallaro R. A. 2003. Presenilin 1 gene silencing by S-adenosylmethionine: a treatment for Alzheimer disease? FEBS Letters 541 (1-3):145-148.    Fuso A., Cavallaro R. A., Orrù L., Buttarelli F. R. and Scarpa S. 2001. Gene silencing by S-adenosylmethionine in muscle differentiation. FEBS Letters 508 (3): 337-340.
The use of SAM to treat AD was also proposed in US 2002/025926 and US 2004/0048827. The latter document demonstrated the ability of SAM to interfere with beta-secretase, presenilin 1 and 2 and beta-amyloid protein precursor gene expression. Studies conducted with labelled (tritiated) SAMe after said patent application demonstrated the ability of the molecule to reach the central nervous system after oral administration, as reported below.
The beneficial effect of SOD in the treatment of AD is suggested in U.S. Pat. No. 5,519,058 and in CN 1099224.
Pharmaceutical compositions comprising SAM and SOD together with a number of active ingredients, for use in conditions other than AD, are disclosed in US 2003/129261 and in WO 2005/041996.